376 FATTY SUBSTANCES (GENERAL METHODS) is kept in a bottle closed with a rubber stopper through which passes a 25 G.C. pipette. (2) Seminormal hydrochloric acid. (3) Alcoholic phenolpMhalein solution (i% in 95% alcohol). PROCEDURE. Inta conical 150-200 c.c. flask, 1-2 grams of the fat are weighed and treated with 25 c.c. of the alcoholic potash solution, the pipette being emptied always in the same way. The flask is closed with a stopper through which passes a glass tube about a metre long to serve as a reflux condenser and heated on a boiling water-bath, with occasional shaking, for half an hour or rather more (see Saponification, 5). The flask is then removed from the bath and the excess of potash remain- ing free titrated with seminormal hydrochloric acid in presence of 8-10 drops of the phenolphthalein solution.1 A check experiment with 25 c.c. of the caustic potash solution alone (without fat) is made at the same time and under the same conditions as the actual test.2 From the difference between the volumes of seminormal hydrochloric acid used in the check experiment and in the actual test with the fat, the amount of potash (milligrams) necessary foi the complete saponification of i gram of the fat is calculated. EXAMPLE : 1-524 gram of a fat, saponified with 25 c.c. of alcoholic potash, required 11-9 c.c. of seminormal hydrochloric acid to neutralise the excess of potash. In the check experiment, 22-5 c.c. of the acid were required for 25 c.c. of alcoholic potash. Since i c.c. N/2-HC1 = 0-02805 gram of KOH, the amount of KOH necessary to saponify 1-524 gram of fat is (22-5-11-9) 0-02805 gram = 0-2973 gram, so that the amount for i gram of fat is 0-195 gram. The saponification number of the fat is hence 195. The saponification number is of importance for distinguishing between different fats and waxes and especially for the analysis of mixtures of fatty substances with non-saponifiable matter (mineral oils, resin oils, etc.). The majority of fatty substances have a saponification number between 190 and 200, but the oils of the Cruciferse (colza oil, ravison oil, etc.), castor oil, grapeseed oil, and a few other oils, have values below 190. Coconut oil, palm-kernel oil, certain other vegetable fats, and butter have numbers above 200. Waxes have very low saponification numbers (below 100). 9. Ester Number The ester number denotes the number of milligrams of caustic potash necessary to saponify the neutral fat (neutral esters) in one gram of a fatty substance. With fats which do not contain free acids, the ester number is equal to the saponification number ; when, however, free acids are present, a little powdered calcium carbonate at such a rate that 50 c.c. pass over in 20 minutes. To test the distilled alcohol, 10 c.c. are boiled with i c.c. of 50 % caustic potash solution and the liquid allowed to stand for 20 minutes to see if any colour develops. If so, the alcohol is unsuitable for preparing alcoholic potash and should be again treated with permanganate. 1 When the fat gives a highly coloured solution with the alcoholic potash, it is advisable to dilute the liquid considerably with neutral alcohol before titrating, in order that the neutral point may be determined with accuracy. 2 Two blank experiments and two actual tests should always be made and the mean of the results taken, provided t hat these do not differ greatly.quid remains clear.r.